The invention comprises antibodies against the human tight junction protein SEMP1, methods for their production and uses thereof in diagnosis and therapy.
The extracellular environment of mammalian cells determines via regulation of cell adhesion and cell proliferation a normal, non-tumorigenic behavior of cells in vivo. Losses of these major control elements are hallmarks of tumorigenesis.
Recently occludin and members of the claudin family have been identified as the major constituents of the cell adhesion complex tight junction. The murine claudin-1 obviously constitutes the major tight junction protein in epithelial cells (Furuse, M., et al., J. Cell Biol. 141 (1998) 1539-1550; Furuse, M., et al., J. Cell Biol. 143 (1998) 391-401), and the human homologue SEMP1 (senescence-associated epithelial membrane protein, Swiss Prot. Acc. No. 095832, CLD1 human) was identified recently by molecular genetic analysis (Swisshelm, K. A., et al., Gene 226 (1999) 285-295). There is ample evidence that tight junction as cellxe2x80x94cell contact and sealing protein might be involved in tumorgenesis (Porvaznik, M., et al., J. Supramol. Struct. 10 (1979) 13-30; Swift, J. G., et al., J. Submicrosc. Cytol. 15 (1983) 799-810; Chochand-Prillet, B., et al., Ultrastruct. Pathol. 22 (1998) 413-420; Soler, A. P., et al., Carcinogenesis 20 (1999) 1425-1431; Woo, P. L., et al., J. Biol. Chem. 274 (1999) 32818-32828). In addition, it has been shown recently that the expression of SEMP1 can be found exclusively in cells and tissue of epithelial origin but it is downregulated or completely lost in human breast cancer tumor cells in vitro (Swisshelm, K. A., et al., Gene 226 (1999) 285-295).
The loss or expression of tight junction proteins or associated molecules in the diagnostic evaluation of tumorigenesis and/or tumor progression or therapeutic intervention in vivo or ex vivo requires polyclonal or monoclonal antibodies. The generation and application of antibodies to identify occludin has been shown successfully in vitro (Furuse, M., et al., J. Cell Biol. 141 (1998) 1539-1550; Furuse, M., et al., J. Cell Biol. 143 (1998) 391-401). However significant difficulties were encountered to generate poly- or monoclonal antibodies against the human SEMP1 or its mouse homologue claudin-1. Disappointing results were also reported to generate monoclonal or polyclonal antibodies against murine claudin-1 (Furuse, M., et al., J. Cell Biol. 141 (1998) 1539-1550).
More recently, one polyclonal antibody was generated in rabbits against the C-terminal intracellular domain of murine claudin-1 protein, which does not cross-react with other related endogenous protein (clone MH25, Zymed Laboratories Inc., 458 Carlton Court, South San Francisco, Calif. 94080, Catalog No. 71-7800, http://www.zymed.com/products/71-7800.html) and one monoclonal antibody was generated against the C-terminal domain of murine claudin-1 in rats (Furuse, M., et al., J. Cell Biol. 147 (1999) 891-903).
The present invention therefore provides polyclonal and monoclonal antibodies which bind to SEMP1 (CLD-1 human) polypeptide in a manner equivalent to an antibody selected from the group consisting of antibodies DSM ACC2458, DSM ACC2459, DSM ACC2461, and DSM ACC2463.
Such an antibody according to the invention does not bind to a considerable extent to the C-terminal intracellular domain of murine claudin-1.
Surprisingly, it was found that though attempts to generate anti-SEMP1 antibodies using SEMP1 polypeptide fragments for immunization failed, it is possible to generate SEMP1 specific antibodies using DNA vaccination, preferably with an additional boost with SEMP1 polypeptide. According to the method of the invention it is now possible to easily provide antibodies against all parts of SEMP1, especially against the extracellular domains, which are useful for modulating signal transducing via SEMP1.
DNA vaccination is possible according to the state of the art. The concept of DNA immunization originated from the observation that naked plasmid DNA injected into muscles of mice resulted in transfection of muscle fibers, expression of the transgene and induction of both a CTL (cytotoxic T cell) and an antibody response (Barry, M. A., et al., Biotechniques 16 (1994) 616-618 and 620; Davis, H. L., et al., Hum. Mol. Genet. 2 (1993) 1847-1851; Davis, H. L., et al., Vaccine 12 (1994) 1503-1509; Davis, H. L., Curr. Opin. Biotechnol. 8 (1997) 635-646; Lowrie, D. B., Nat. Med. 4 (1998) 147-148; Ulivieri, C., et al., J. Biotechnol. 51 (1996) 191-194).
The constructs used for DNA immunization are identical to the ones used for delivery of reporter or therapeutic genes. Basically, any of the established eukaryotic expression vectors can be used. Most DNA immunization vectors comprise a strong viral promoter/enhancer sequence to drive high levels of transgene expression in a wide variety of host cells. Also, a polyadenylation sequence to terminate the expressed RNA is required.